Methods for enhancing hematopoietic stem/progenitor cell engraftment

ABSTRACT

Described herein are methods for enhancing engraftment of hematopoietic stem and progenitor cells using farnesyl compounds identified using a zebrafish model of hematopoietic cell engraftment. The compounds can be used to treat hematopoietic stem cells ex vivo prior to transplantation of the cells. Alternatively, the compounds can be administered to an individual undergoing cell transplantation.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a Continuation Application of U.S. Ser. No.13/147,710 filed Aug. 19, 2011, which is a 35 U.S.C. §371 National PhaseEntry Application of International Application No. PCT/US2010/022998filed Feb. 3, 2010, which designates the U.S., and which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/149,499, filed Feb. 3, 2009, and U.S. Provisional Application No.61/186,929, filed Jun. 15, 2009, the contents of which are incorporatedherein by reference in their entireties.

FIELD OF THE INVENTION

The field of the invention relates to enhancing hematopoietic stem celland progenitor cell engraftment following transplantation.

BACKGROUND

Hematopoietic stem and progenitor cell transplantation is used for thetreatment of a wide variety of hematologic disorders, malignancies, andgenetic diseases of the blood. For example, hematopoietic progenitorcell transplantation is currently used to treat bone marrow destructioncaused by irradiation and/or alkylating therapy in the treatment ofcancer.

Hematopoietic progenitor cells are responsible for hematopoieticrecovery during the early post-transplant period. However, in some casesprogenitor cell engraftment fails to occur due to e.g.,micro-environmental defects as part of the underlying disease (e.g.,aplastic anemia), stromal cell damage caused by chemoradiotherapy anddevelopment of graft-versus-host disease. In some cases, long-termengraftment and hematopoietic recovery also fails to occur. The failureof long-term engraftment to occur is attributed to the lack of cellularengraftment of hematopoietic stem cells.

Hematopoietic stem cells are capable of self-renewal and as such areresponsible for maintaining engraftment over prolonged periods of time(e.g., “long-term engraftment”).

Thus, there is a need for the development of methods that improveengraftment of hematopoietic progenitor cells and hematopoietic stemcells.

SUMMARY OF THE INVENTION

Described herein are methods for enhancing engraftment of hematopoieticstem and progenitor cells using farnesyl compounds, such as e.g.,S-farnesyl-L-cysteine methyl ester (FCME), farnesylthioacetic acid(FTA), 2-(farnesylthio)benzoic acid (farnesyl-thiosalicylic acid, FTS),2-chloro-5-farnesylaminobenzoic acid (NFCB),3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinicacid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate, andfarnesyl pyrophosphate (FPP). The compounds can be used to treathematopoietic stem cells ex vivo prior to transplantation of the cells.Alternatively, the compounds can be administered to an individualundergoing cell transplantation.

Provided herein are methods for enhancing hematopoietic cell engraftmentin a subject, the method comprising: (a) contacting a population ofhematopoietic cells with a compound selected from the group consistingof: S-farnesyl-L-cysteine methyl ester (FCME), farnesylthioacetic acid(FTA), 2-(farnesylthio)benzoic acid (farnesyl-thiosalicylic acid, FTS),2-chloro-5-farnesylaminobenzoic acid (NFCB),3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinicacid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate, andfarnesyl pyrophosphate (FPP).

In one aspect, the compound is S-farnesyl-L-cysteine methyl ester (FCME)or a derivative thereof.

In one embodiment of this aspect and all other aspects described herein,the compound has a structure of

In another aspect, the compound is farnesylthioacetic acid (FTA) or aderivative thereof.

In one embodiment of this aspect and all other aspects described herein,the compound has a structure of

In other embodiments, the farnesyl compounds can include, but are notlimited to, 2-(farnesylthio)benzoic acid (farnesyl-thiosalicylic acid,FTS), 2-chloro-5-farnesylaminobenzoic acid (NFCB),3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinicacid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate,farnesyl pyrophosphate (FPP), and those described in U.S. Pat. No.5,705,528; 5,475,029; European patent No. 356,866; Philips, et. al.,Science (1993), 259: 977-980; Akbar, et al., Proc. Natl. Acad. Sci. USA(1993), 90: 868-872; Tan, et al., J. Biol. Chem., (1991), 26(6):10719-10722 and U.S. Pat. No. 5,705,528.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from cordblood.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from bonemarrow.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from blood.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells are isolated cells.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is a heterogeneous orhomogeneous population of cells.

In another embodiment of this aspect and all other aspects describedherein, the contacting step is performed on ex vivo cells in culture.

In another embodiment of this aspect and all other aspects describedherein, wherein the subject is a human subject.

In another embodiment of this aspect and all other aspects describedherein, wherein the hematopoietic cells are hematopoietic stem cells.

In another embodiment of this aspect and all other aspects describedherein, the hematopoietic cells are hematopoietic progenitor cells.

Another aspect described herein relates to a method for enhancinghematopoietic cell engraftment in a subject following hematopoietic celltransplantation, the method comprising: administering to a subjectfollowing hematopoietic cell transplantation a therapeutically effectiveamount of a compound selected from the group consisting of:S-farnesyl-L-cysteine methyl ester (FCME), farnesylthioacetic acid(FTA), 2-(farnesylthio)benzoic acid (farnesyl-thiosalicylic acid, FTS),2-chloro-5-farnesylaminobenzoic acid (NFCB),3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinicacid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate, andfarnesyl pyrophosphate (FPP)

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from cordblood.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from bonemarrow.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is derived from blood.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells are isolated cells.

In another embodiment of this aspect and all other aspects describedherein, the population of hematopoietic cells is a heterogeneous orhomogeneous population of cells.

In another embodiment of this aspect and all other aspects describedherein, the subject is a human subject.

In another embodiment of this aspect and all other aspects describedherein, the hematopoietic cells are hematopoietic stem cells.

In another embodiment of this aspect and all other aspects describedherein, the hematopoietic cells are hematopoietic progenitor cells.

In another aspect, an admixture of hematopoietic stem cells and at leastone compound as described herein is administered to a subjectsimultaneously.

DEFINITIONS

As used herein, by a “subject” is meant an individual. Thus, subjectsinclude, for example, domesticated animals, such as cats and dogs,livestock (e.g., cattle, horses, pigs, sheep, and goats), laboratoryanimals (e.g., mice, rabbits, rats, and guinea pigs) mammals, non-humanmammals, primates, non-human primates, rodents, birds, reptiles,amphibians, fish, and any other animal. The subject is optionally amammal such as a primate or a human.

The term “engraftment” is used herein to refer to the ability ofhematopoietic stem cells or progenitor cells to repopulate a tissue,whether such cells are naturally circulating or are provided bytransplantation. The term encompasses all events surrounding or leadingup to engraftment, such as tissue homing of cells and colonization ofcells within the tissue of interest. The engraftment efficiency or rateof engraftment can be evaluated or quantified using any clinicallyacceptable parameter as known to those of skill in the art and caninclude, for example, assessment of competitive repopulating units(CRU); incorporation or expression of a marker in tissue(s) into whichstem cells have homed, colonized, or become engrafted; or by evaluationof the progress of a subject through disease progression, survival ofhematopoietic progenitor cells, or survival of a recipient. In oneembodiment, engraftment is determined by measuring white blood cellcounts in peripheral blood during a post-transplant period.Alternatively, engraftment can be assessed by measuring recovery ofmarrow cells in a bone marrow aspirate sample.

As used herein, the term “enhancing hematopoietic cell engraftment”refers to an increase in the efficiency or rate (i.e., amount ofengraftment over a period of time) of hematopoietic progenitor cell orstem cell engraftment of at least 10% (e.g., as assessed by measuringwhite blood cell count) in individuals treated with a compound (or in anindividual administered cells treated with an agent) compared tountreated individuals. Preferably the rate of hematopoietic cellengraftment is increased by at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 99%, at least 1-fold, at least 2-fold, at least5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least1000-fold or higher in individuals being treated with an agent comparedto the efficiency/rate of engraftment in an untreated individual.Engraftment can also be assessed using a bone marrow aspirate sample andmonitoring colony forming unit cells (CFU-Cs).

As used herein, the term “hematopoietic progenitor cells” encompassespluripotent cells capable of differentiating into several cell types ofthe hematopoietic system, including, but not limited to, granulocytes,monocytes, erythrocytes, megakaryocytes, B-cells and T-cells.Hematopoietic progenitor cells are committed to the hematopoietic celllineage and generally do not self-renew; hematopoietic progenitor cellscan be identified, for example by cell surface markers such asLin−KLS+Flk2−CD34+. The term “hematopoietic progenitor cells”encompasses short term hematopoietic stem cells (ST-HSCs), multi-potentprogenitor cells (MPPs), common myeloid progenitor cells (CMPs),granulocyte-monocyte progenitor cells (GMPs), andmegakaryocyte-erythrocyte progenitor cells (MEPs). The term“hematopoietic progenitor cells” does not encompass hematopoietic stemcells capable of self-renewal (herein referred to as “hematopoietic stemcells”), which can be identified with the following stem cell markerprofile: Lin−KLS+Flk2−CD34−. The presence of hematopoietic progenitorcells can be determined functionally as colony forming unit cells(CFU-Cs) in complete methylcellulose assays, or phenotypically throughthe detection of cell surface markers using assays known to those ofskill in the art.

As used herein, the term “hematopoietic stem cell (HSC)” refers to acell with multi-lineage hematopoietic differentiation potential andsustained self-renewal activity. “Self renewal” refers to the ability ofa cell to divide and generate at least one daughter cell with theidentical (e.g., self-renewing) characteristics of the parent cell. Thesecond daughter cell may commit to a particular differentiation pathway.For example, a self-renewing hematopoietic stem cell divides and formsone daughter stem cell and another daughter cell committed todifferentiation in the myeloid or lymphoid pathway. A committedprogenitor cell has typically lost the self-renewal capacity, and uponcell division produces two daughter cells that display a moredifferentiated (i.e., restricted) phenotype. Hematopoietic stem cellshave the ability to regenerate long term multi-lineage hematopoiesis(e.g., “long-term engraftment”) in individuals receiving a bone marrowor cord blood transplant. The hematopoietic stem cells used may bederived from any one or more of the following sources: fetal tissues,cord blood, bone marrow, peripheral blood, mobilized peripheral blood, astem cell line, or may be derived ex vivo from other cells, such asembryonic stem cells, induced pluripotent stem cells (iPS cells) oradult pluripotent cells. The cells from the above listed sources may beexpanded ex vivo using any method acceptable to those skilled in the artprior to use in the transplantation procedure. For example, cells may besorted, fractionated, treated to remove malignant cells, or otherwisemanipulated to treat the patient using any procedure acceptable to thoseskilled in the art of preparing cells for transplantation. If the cellsused are derived from an immortalized stem cell line, further advantageswould be realized in the ease of obtaining and preparation of cells inadequate quantities.

As used herein, the term “population of hematopoietic cells” encompassesa heterogeneous or homogeneous population of hematopoietic stem cellsand/or hematopoietic progenitor cells. In addition, differentiatedhematopoietic cells, such as white blood cells, can be present in apopulation of hematopoietic cells; that is, in one embodimenthematopoietic stem and/or progenitor cells are not isolated from e.g.,cord blood, bone marrow. A population of hematopoietic cells comprisingat least two different cell types is referred to herein as a“heterogeneous population”. It is also contemplated herein thathematopoietic stem cells or hematopoietic progenitor cells are isolatedand expanded ex vivo prior to transplantation. A population ofhematopoietic cells comprising only one cell type (e.g., hematopoieticstem cells) is referred to herein as a “homogeneous population ofcells”.

“Expansion” or “expanded” in the context of cells refers to an increasein the number of a characteristic cell type, or cell types, from aninitial population of cells, which may or may not be identical. Theinitial cells used for expansion need not be the same as the cellsgenerated from expansion. For instance, the expanded cells may beproduced by ex vivo or in vitro growth and differentiation of theinitial population of cells. It is contemplated herein that ahematopoietic stem cell or progenitor cell is expanded in culture priorto transplantation into an individual in need thereof.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

As used herein the term “consisting essentially of” refers to thoseelements required for a given embodiment. The term permits the presenceof elements that do not materially affect the basic and novel orfunctional characteristic(s) of that embodiment of the invention.

The term “consisting of” refers to compositions, methods, and respectivecomponents thereof as described herein, which are exclusive of anyelement not recited in that description of the embodiment.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural references unless the contextclearly dictates otherwise. Thus for example, references to “the method”includes one or more methods, and/or steps of the type described hereinand/or which will become apparent to those persons skilled in the artupon reading this disclosure and so forth.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1C show a schematic representation of the design of anexemplary adult zebrafish competitive transplantation assay. 1A) Flowchart of a competitive transplantation in adult zebrafish (casper fishas recipients); 1B) quantification of relative engraftment by imagefluorescence analysis. G/R, GFP intensity/DsRed2 intensity; Gkid, kidneyGFP intensity; Gbkg, background GFP intensity; Rkid, kidney DsRed2intensity; Rbkg, background DsRed2 intensity. 1C) positive correlationof FACS analysis results and image fluorescence analysis results. TheG/R of FACS analysis is defined by the ratio of the percentage of GFP+cells over the percentage of DsRed2+ cells.

FIGS. 2A-2C show data from a competitive transplantation assay thatfaithfully represents the donor ratio. 2A) is a schematic depicting thecompetitive transplant protocol. 2B) is a table indicating an increasein the green:red fluorescence ratio with increasing numbers of treateddonor cells. 2C) is a graph depicting the data from FIG. 2B.

FIGS. 3A-3C shows the effect of positive control drugs on engraftment inthe competitive transplantation assay. 3A) is a schematic of a protocolfor a competitive transplant assay using drug treated cells. 3B) is atable representing fluorescence data from zebrafish transplanted withvehicle treated (DMSO) or drug treated cells. 3C) is a graph depictingthe data from FIG. 3B.

FIG. 4 is a schematic flow chart depicting a chemical screen using theadult zebrafish competitive marrow transplantation assay.

FIG. 5 is a graph showing competitive transplantation with differentdrug treatments for the GFP+ marrow.

FIGS. 6A and 6B are each a graph that shows fluorescence data fortransplanted zebrafish with cells treated with compounds from anexemplary chemical screen at both 2 wpt (6A) and 4 wpt (6B).

FIGS. 7A and 7B are each a bar graph depicting data representing a mousecompetitive transplant assay that is used to confirm the accuracy of thezebrafish assay described herein.

DETAILED DESCRIPTION

The methods described herein are based, in part, on the discovery of aclass of compounds that enhance engraftment of hematopoietic stem and/orprogenitor cells following transplantation, such as a bone marrow orcord blood transplant. Accordingly, provided herein are methods forenhancing engraftment of hematopoietic stem and/or progenitor cells bytreating cells ex vivo with a farnesyl compound prior to transplantationto an individual in need thereof. Also provided herein are methods forenhancing engraftment comprising administering a farnesyl compound asdescribed herein to an individual undergoing a hematopoietic stem and/orprogenitor cell transplant.

Hematopoietic Stem/Progenitor Cells

Hematopoietic Stem Cells

Hematopoietic stem cells (HSC) are primitive cells capable ofregenerating all blood cells. During development, the site ofhematopoiesis translocates from the fetal liver to the bone marrow,which then remains the site of hematopoiesis throughout adulthood.

HSC as used herein refer to immature blood cells having the capacity toself-renew and to differentiate into more mature blood cells comprisinggranulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils),erythrocytes (e.g., reticulocytes, erythrocytes), thrombocytes (e.g.,megakaryoblasts, platelet producing megakaryocytes, platelets), andmonocytes (e.g., monocytes, macrophages). Hematopoietic stem cells areinterchangeably described as stem cells throughout the specification. Itis known in the art that such cells may or may not include CD34+ cells.CD34+ cells are immature cells that express the CD34 cell surfacemarker. CD34+ cells are believed to include a subpopulation of cellswith the stem cell properties defined above. It is well known in the artthat hematopoietic stem cells include pluripotent stem cells,multipotent stem cells (e.g., a lymphoid stem cell), and/or stem cellscommitted to specific hematopoietic lineages. The stem cells committedto specific hematopoietic lineages may be of T cell lineage, B celllineage, dendritic cell lineage, Langerhans cell lineage and/or lymphoidtissue-specific macrophage cell lineage. In addition, HSCs also refer tolong term HSC (LT-HSC) and short term HSC (ST-HSC). A long term stemcell typically includes the long term (more than three months)contribution to multilineage engraftment after transplantation. A shortterm stem cell is typically anything that lasts shorter than threemonths, and/or that is not multilineage. LT-HSC and ST-HSC aredifferentiated, for example, based on their cell surface markerexpression. LT-HSC are CD34−, SCA-1+, Thy.1.1+/lo, C-kit+, Un−, CD135−,Slamfl/CD150+, whereas ST-HSC are CD34+, SCA-1+, Thy.1.1+/lo, C-kit+,lin−, CD135−, Slamfl/CD150+, Mac-1 (CD1Ib)lo (“lo” refers to lowexpression). In addition, ST-HSC are less quiescent (i.e., more active)and more proliferative than LT-HSC. LT-HSC have unlimited self renewal(i.e., they survive throughout adulthood), whereas ST-HSC have limitedself renewal (i.e., they survive for only a limited period of time). Anyof these HSCs can be used in any of the methods described herein.

HSC are optionally obtained from blood products. A blood productincludes a product obtained from the body or an organ of the bodycontaining cells of hematopoietic origin. Such sources includeunfractionated bone marrow, umbilical cord, peripheral blood, liver,thymus, lymph and spleen. All of the aforementioned crude orunfractionated blood products can be enriched for cells havinghematopoietic stem cell characteristics in a number of ways. Forexample, the more mature, differentiated cells are selected against, viacell surface molecules they express. Optionally, the blood product isfractionated by selecting for CD34+ cells. CD34+ cells include asubpopulation of cells capable of self-renewal and pluripotentiality.Such selection is accomplished using, for example, commerciallyavailable magnetic anti-CD34 beads (Dynal, Lake Success, N.Y.).Unfractionated blood products are optionally obtained directly from adonor or retrieved from cryopreservative storage.

Sources for HSC expansion also include aorta-gonad-mesonephros (AGM)derived cells, embryonic stem cell (ESC) and induced pluripotent stemcells (iPSC). ESC are well-known in the art, and may be obtained fromcommercial or academic sources (Thomson et al., 282 Sci. 1145-47(1998)). iPSC are a type of pluripotent stem cell artificially derivedfrom a non-pluripotent cell, typically an adult somatic cell, byinducing a “forced” expression of certain genes (Baker, Nature Rep. StemCells (Dec. 6, 2007); Vogel & Holden, 23 Sci. 1224-25 (2007)). ESC, AGM,and iPSC may be derived from animal or human sources. The AGM stem cellis a cell that is born inside the aorta, and colonizes the fetal liver.Signaling pathways can increase AGM stem cells make it likely that thesepathways will increase HSC in ESC.

Hematopoietic Progenitor Cells

Hematopoietic progenitor cells, as the term is used herein, are capableof producing all cells types in the hematopoietic lineage, but are notcapable of long-term self-renewal. Thus, hematopoietic progenitor cellscan restore and sustain hematopoiesis for three to four months (Marshak,D. R., et al. (2001). Stem cell biology, Cold Spring Harbor, N.Y.: ColdSpring Harbor Laboratory Press) and are important for recovery in theperiod immediately following a hematopoietic progenitor cell transplantin an individual. Hematopoietic progenitor cells useful fortransplantation can be obtained from a variety of sources including, forexample, bone marrow, peripheral blood, and umbilical cord blood.

Bone marrow can be obtained by puncturing bone with a needle andremoving bone marrow cells with a syringe (herein called “bone marrowaspirate”). Hematopoietic progenitor cells can be isolated from the bonemarrow aspirate prior to transplantation by using surface markersspecific for hematopoietic progenitor cells, or alternatively whole bonemarrow can be transplanted into an individual to be treated with themethods described herein.

Hematopoietic progenitor cells can also be obtained from peripheralblood of a progenitor cell donor. Prior to harvest of the cells fromperipheral blood, the donor can be treated with a cytokine, such ase.g., granulocyte-colony stimulating factor, to promote cell migrationfrom the bone marrow to the blood compartment. Cells can be collectedvia an intravenous tube and filtered to isolate white blood cells fortransplantation. The white blood cell population obtained (i.e., amixture of stem cells, progenitors and white blood cells of variousdegrees of maturity) can be transplanted as a heterogeneous mixture orhematopoietic progenitor cells can further be isolated using cellsurface markers known to those of skill in the art.

Hematopoietic progenitor cells and/or a heterogeneous hematopoieticprogenitor cell population can also be isolated from human umbilicalcord and/or placental blood.

Isolation of Hematopoietic Stem Cells

A hematopoietic stem cell (HSC) population can be obtained from a biopsyremoved from a donor employing techniques known by persons skilled inthe art, including the removal of stem cells from the bone marrow of adonor from large bone masses utilizing a large needle intended for bonemarrow harvesting. Alternatively, HSCs may be collected by apheresis, aprocess in which a donor's peripheral blood is withdrawn through asterile needle and passed through a device that removes white bloodcells, and that returns the red blood cells to the donor. The peripheralstem cell yield can be increased with daily subcutaneous injections ofgranulocyte-colony stimulating factor. The HSCs are preferably obtainedfrom human donors, however, non-human donors are also contemplated,including non-human primates, pigs, cows, horses, cats, and dogs. Apurified population of HSCs may be obtained by utilizing various methodsknown by persons skilled in the art and described in U.S. Pat. No.5,677,136; and U.S. Patent Publication No. 2006/0040389, which areincorporated by reference in their entirety.

In one embodiment described herein, hematopoietic stem cells orprogenitor cells are isolated prior to transplantation. Hematopoieticcell samples (e.g., cord blood, peripheral blood, bone marrow) can firstbe purified to isolate and obtain artificially high concentrations ofe.g., HSCs by detecting expression of specific cell surface proteins orreceptors, cell surface protein markers, or other markers. Highlypurified HSCs are increasingly being used clinically, in a variety ofapplications, such as for autologous transplants into patients afterhigh-dose chemotherapy. In this setting it is advantageous to isolateHSCs with the maximum degree of purity so as to minimize contaminationby immune effector cells (such as lymphocytes) or cancer cells. Inmurine studies, the highest enrichment of HSC activity yet reporteddescribes combinations of markers, such as those used to isolateThy-1.1^(lo)Sca-1⁺lineage-Mac-1-CD4⁻c-kit⁺ cells, from which about oneout of every five intravenously injected cells are able to home to bonemarrow and engraft. Such results are described in, for example, Uchidaet al.; Morrison et al., 1994 and Morrison et al. 1997 supra).

Stem cells as well as committed progenitor cells destined to becomeneutrophils, erythrocytes, platelets, etc., may be distinguished frommost other cells by the presence or absence of particular progenitormarker antigens, such as CD34, that are present on the surface of thesecells and/or by morphological characteristics. The phenotype for ahighly enriched human stem cell fraction is reported as CD34⁺,Thy-1⁺ andlin⁻, but it is to be understood that the present invention is notlimited to the expansion of this stem cell population.

The CD34+ enriched human stem cell fraction can be separated by a numberof reported methods, including affinity columns or beads, magnetic beadsor flow cytometry using antibodies directed to surface antigens such asthe CD34+. Further, physical separation methods such as counterflowelutriation may be used to enrich hematopoietic progenitors. The CD34+progenitors are heterogeneous, and may be divided into severalsubpopulations characterized by the presence or absence of coexpressionof different lineage associated cell surface associated molecules. Themost immature progenitor cells do not express any knownlineage-associated markers, such as HLA-DR or CD38, but they may expressCD90 (thy-1). Other surface antigens such as CD33, CD38, CD41, CD71,HLA-DR or c-kit can also be used to selectively isolate hematopoieticprogenitors. The separated cells can be incubated in selected medium ina culture flask, sterile bag or in hollow fibers. Various hematopoieticgrowth factors may be utilized in order to selectively expand cells.Representative factors that have been utilized for ex vivo expansion ofbone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6,IL-11, flt-3 ligand or combinations thereof. The proliferation of stemcells can be monitored by enumerating the number of stem cells and othercells, by standard techniques (e. g., hemacytometer, CFU, LTCIC) or byflow cytometry prior and subsequent to incubation.

Any method suitable for identifying surface proteins, whether known orto be discovered, could be employed to isolate hematopoietic stem cellsfrom a homogeneous population such as e.g., cord blood. For example,HSCs for use with the methods described herein may be identified usingfluorescence activated cell sorting analysis (FACS) which typically usesantibodies conjugated to fluorochromes to directly or indirectly assessthe level of expression of a given surface protein on individual cellswithin a heterogenous (or homogenous) cell preparation of hematopoietictissue.

HSCs may be physically separated from other cells within a cellularpreparation of hematopoietic tissue using any previously developed or asyet undeveloped technique whereby cells are directly or indirectlydifferentiated according to their expression or lack of expression ofparticular surface proteins. Common methods used to physically separatespecific cells from within a heterogenous population of cells within ahematopoietic cell preparation include but are not limited toflow-cytometry using a cytometer which may have varying degrees ofcomplexity and or detection specifications, magnetic separation, usingantibody or protein coated beads, affinity chromatography, orsolid-support affinity separation where cells are retained on asubstrate according to their expression or lack of expression of aspecific protein or type of protein. Such separation techniques neednot, but may, completely purify or nearly completely purify (e.g. 99.9%are perfectly separated) HSCs or populations enriched in HSCs.

Cell Culture and Expansion of Isolated Hematopoietic Stem/ProgenitorCells

The expanded population of stem cells are harvested, for example, from abone marrow sample of a subject or from a culture. Harvestinghematopoietic stem cells is defined as the dislodging or separation ofcells. This is accomplished using a number of methods, such asenzymatic, non-enzymatic, centrifugal, electrical, or size-basedmethods, or preferably, by flushing the cells using culture media (e.g.,media in which cells are incubated) or buffered solution. The cells areoptionally collected, separated, and further expanded generating evenlarger populations of HSC and differentiated progeny.

In general, cells useful for the invention can be maintained andexpanded in culture medium that is available to and well-known in theart. Such media include, but are not limited to, Dulbecco's ModifiedEagle's Medium® (DMEM), DMEM F12 Medium®, Eagle's Minimum EssentialMedium®, F-12K Medium®, Iscove's Modified Dulbecco's Medium®, RPMI-1640Medium®, and serum-free medium for culture and expansion ofhematopoietic cells SFEM®. Many media are also available as low-glucoseformulations, with or without sodium pyruvate.

Also contemplated herein is supplementation of cell culture medium withmammalian sera. Sera often contain cellular factors and components thatare necessary for viability and expansion. Examples of sera includefetal bovine serum (FBS), bovine serum (BS), calf serum (CS), fetal calfserum (FCS), newborn calf serum (NCS), goat serum (GS), horse serum(HS), human serum, chicken serum, porcine serum, sheep serum, rabbitserum, serum replacements and bovine embryonic fluid. It is understoodthat sera can be heat-inactivated at 55-65° C. if deemed necessary toinactivate components of the complement cascade.

Additional supplements also can be used advantageously to supply thecells with the necessary trace elements for optimal growth andexpansion. Such supplements include insulin, transferrin, sodiumselenium and combinations thereof. These components can be included in asalt solution such as, but not limited to, Hanks' Balanced SaltSolution® (HBSS), Earle's Salt Solution®, antioxidant supplements,MCDB-201® supplements, phosphate buffered saline (PBS), ascorbic acidand ascorbic acid-2-phosphate, as well as additional amino acids. Manycell culture media already contain amino acids, however, some requiresupplementation prior to culturing cells. Such amino acids include, butare not limited to, L-alanine, L-arginine, L-aspartic acid,L-asparagine, L-cysteine, L-cystine, L-glutamic acid, L-glutamine,L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan,L-tyrosine, and L-valine. It is well within the skill of one in the artto determine the proper concentrations of these supplements.

Hormones also can be advantageously used in the cell cultures of thepresent invention and include, but are not limited to, D-aldosterone,diethylstilbestrol (DES), dexamethasone, .beta.-estradiol,hydrocortisone, insulin, prolactin, progesterone, somatostatin/humangrowth hormone (HGH), thyrotropin, thyroxine and L-thyronine.

Lipids and lipid carriers also can be used to supplement cell culturemedia, depending on the type of cell and the fate of the differentiatedcell. Such lipids and carriers can include, but are not limited to,cyclodextrin (α, β, γ), cholesterol, linoleic acid conjugated toalbumin, linoleic acid and oleic acid conjugated to albumin,unconjugated linoleic acid, linoleic-oleic-arachidonic acid conjugatedto albumin and oleic acid unconjugated and conjugated to albumin, amongothers.

Also contemplated in the present invention is the use of feeder celllayers. Feeder cells are used to support the growth of fastidiouscultured cells, such as stem cells. Feeder cells are normal cells thathave been inactivated by y-irradiation. In culture, the feeder layerserves as a basal layer for other cells and supplies cellular factorswithout further growth or division of their own (Lim, J. W. and Bodnar,A., 2002). Examples of feeder layer cells are typically human diploidlung cells, mouse embryonic fibroblasts and Swiss mouse embryonicfibroblasts, but can be any post-mitotic cell that is capable ofsupplying cellular components and factors that are advantageous inallowing optimal growth, viability and expansion of stem cells. In manycases, feeder cell layers are not necessary to keep stem cells in anundifferentiated, proliferative state, as leukemia inhibitory factor(LIF) has anti-differentiation properties. Therefore, supplementationwith LIF can be used to maintain cells in an undifferentiated state.

Cells may be cultured in low-serum or serum-free culture medium.Serum-free medium used to culture cells is described in, for example,U.S. Pat. No. 7,015,037. Many cells have been grown in serum-free orlow-serum medium. For example, the medium can be supplemented with oneor more growth factors. Commonly used growth factors include, but arenot limited to, bone morphogenic protein, basic fibroblast growthfactor, platelet-derived growth factor and epidermal growth factor, Stemcell factor, thrombopoietin, Flt3Ligand and 1′-3. See, for example, U.S.Pat. Nos. 7,169,610; 7,109,032; 7,037,721; 6,617,161; 6,617,159;6,372,210; 6,224,860; 6,037,174; 5,908,782; 5,766,951; 5,397,706; and4,657,866; all incorporated by reference herein for teaching growingcells in serum-free medium.

Cells in culture can be maintained either in suspension or attached to asolid support, such as extracellular matrix components. Stem cells oftenrequire additional factors that encourage their attachment to a solidsupport, such as type I and type II collagen, chondroitin sulfate,fibronectin, “superfibronectin” and fibronectin-like polymers, gelatin,poly-D and poly-L-lysine, thrombospondin and vitronectin. Hematopoieticstem cells can also be cultured in low attachment flasks such as but notlimited to Corning Low attachment plates.

In one embodiment, hematopoietic stem and/or progenitor cells aretreated ex vivo prior to transplantation to an individual in needthereof by contacting a population of hematopoetic cells with a farnesylcompound. Contacting can be performed in vitro by adding the farnesylcompound directly to suitable cell culture medium for hematopoieticcells. The concentration of compound can be determined by those of skillin the art, for example by performing serial dilutions and testingefficacy in the Zebrafish competitive transplant model, or othersuitable system. Example concentration ranges for the treatment of thehematopoietic stem and/or progenitor cells include, but are not limitedto, about 1 nanomolar to about 10 millimolar; about 1 mM to about 5 mM;about 1 nM to about 500 nM; about 500 nM to about 1,000 nM; about 1 nMto about 1,000 nM; about 1 uM to about 1,000 uM; 1 uM to about 500 uM;about 1 uM to about 100 uM; about 1 uM to about 10 uM. In oneembodiment, the range is about 5 uM to about 500 uM.

Cells can be treated for various times. Suitable times can be determinedby those of skill in the art. For example, cells can be treated forminutes, 15 minutes, 30 minutes etc, or treated for hours e.g., 1 hour,2 hours, 3 hours, 4 hours, up to 24 hours or even days. In oneembodiment the cells are treated for 2 hours prior to changing to mediumwithout drug.

Cryopreservation of Cells and Blood

Once established in culture, cells treated with the farnesyl compounds(e.g. FTA, or FCME), and/or untreated cells can be used fresh or frozenand stored as frozen stocks, using, for example, DMEM with 40% FCS and10% DMSO. Other methods for preparing frozen stocks for cultured cellsalso are available to those skilled in the art.

In addition, the stem cells obtained from harvesting according to methodof the present invention described above can be cryopreserved usingtechniques known in the art for stem cell cryopreservation. Accordingly,using cryopreservation, the stem cells can be maintained such that onceit is determined that a subject is in need of stem cell transplantation,the stem cells can be thawed and transplanted back into the subject.

More specifically, an embodiment of the present invention provides forthe enhancement of HSCs collected from cord blood or an equivalentneonatal or fetal stem cell source, which may be cryopreserved, for thetherapeutic uses of such stem cells upon thawing. Such blood may becollected by several methods known in the art. For example, becauseumbilical cord blood is a rich source of HSCs (see Nakahata & Ogawa, 70J. Clin. Invest. 1324-28 (1982); Prindull et al., 67 Acta. Paediatr.Scand. 413-16 (1978); Tchernia et al., 97(3) J. Lab. Clin. Med. 322-31(1981)), an excellent source for neonatal blood is the umbilical cordand placenta. Prior to cryopreservation, the neonatal blood may beobtained by direct drainage from the cord and/or by needle aspirationfrom the delivered placenta at the root and at distended veins. See,e.g., U.S. Pat. Nos. 7,160,714; No. 5,114,672; No. 5,004,681; U.S.patent application Ser. No. 10/076,180, Pub. No. 20030032179.Indeed,umbilical cord blood stem cells have been used to reconstitutehematopoiesis in children with malignant and nonmalignant diseases aftertreatment with myeloablative doses of chemo-radiotherapy. Sirchia &Rebulla, 84 Haematologica 738-47 (1999). See also Laughlin 27 BoneMarrow Transplant. 1-6 (2001); U.S. Pat. No. 6,852,534. Additionally, ithas been reported that stem and progenitor cells in cord blood appear tohave a greater proliferative capacity in culture than those in adultbone marrow. Salahuddin et al., 58 Blood 931-38 (1981); Cappellini etal., 57 Brit. J. Haematol. 61-70 (1984).

Alternatively, fetal blood can be taken from the fetal circulation atthe placental root with the use of a needle guided by ultrasound (Daffoset al., 153 Am. J. Obstet. Gynecol. 655-60 (1985); Daffos et al., 146Am. J. Obstet. Gynecol. 985-87 (1983), by placentocentesis (Valenti, 115Am. J. Obstet. Gynecol. 851-53 (1973); Cao et al., 19 J. Med. Genet.81-87 (1982)), by fetoscopy (Rodeck, in PRENATAL DIAGNOSIS, (Rodeck &Nicolaides, eds., Royal College of Obstetricians & Gynaecologists,London, 1984)) and cryopreserved. Indeed, the chorionic villus andamniotic fluid, in addition to cord blood and placenta, are sources ofpluripotent fetal stem cells (see WO 2003 042405).

Various kits and collection devices are known for the collection,processing, and storage of cord blood. See, e.g., U.S. Pat. Nos.7,147,626; No. 7,131,958. Collections should be made under sterileconditions, and the blood may be treated with an anticoagulant. Suchanticoagulants include citrate-phosphate-dextrose, acidcitrate-dextrose, Alsever's solution (Alsever & Ainslie, 41 N.Y. St. J.Med. 126-35 (1941), DeGowin's Solution (DeGowin et al., 114 JAMA 850-55(1940)), Edglugate-Mg (Smith et al., 38 J. Thorac. Cardiovasc. Surg.573-85 (1959)), Rous-Turner Solution (Rous & Turner, 23 J. Exp. Med.219-37 (1916)), other glucose mixtures, heparin, or ethylbiscoumacetate. See Hurn, STORAGE OF BLOOD 26-160 (Acad. Press, NY,1968).

Various procedures are known in the art or described herein and can beused to enrich collected cord blood for HSCs. These include but are notlimited to equilibrium density centrifugation, velocity sedimentation atunit gravity, immune rosetting and immune adherence, counterflowcentrifugal elutriation, T-lymphocyte depletion, andfluorescence-activated cell sorting, alone or in combination. See, e.g.,U.S. Pat. No. 5,004,681.

Typically, collected blood is prepared for cryogenic storage by additionof cryoprotective agents such as DMSO (Lovelock & Bishop, 183 Nature1394-95 (1959); Ashwood-Smith 190 Nature 1204-05 (1961)), glycerol,polyvinylpyrrolidine (Rinfret, 85 Ann. N.Y. Acad. Sci. 576-94 (1960)),polyethylene glycol (Sloviter & Ravdin, 196 Nature 899-900 (1962)),albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol,D-mannitol (Rowe, 3(1) Cryobiology 12-18 (1966)), D-sorbitol,i-inositol, D-lactose, choline chloride (Bender et al., 15 J. Appl.Physiol. 520-24 (1960)), amino acids (Phan & Bender, 20 Exp. Cell Res.651-54 (1960)), methanol, acetamide, glycerol monoacetate (Lovelock, 56Biochem. J. 265-70 (1954)), and inorganic salts (Phan & Bender, 104Proc. Soc. Exp. Biol. Med. (1960)). Addition of plasma (e.g., to aconcentration of 20%-25%) may augment the protective effect of DMSO.

Collected blood should be cooled at a controlled rate for cryogenicstorage. Different cryoprotective agents and different cell types havedifferent optimal cooling rates. See e.g., Rapatz, 5 Cryobiology 18-25(1968), Rowe & Rinfret, 20 Blood 636-37 (1962); Rowe, 3 Cryobiology12-18 (1966); Lewis et al., 7 Transfusion 17-32 (1967); Mazur, 168Science 939-49 (1970). Considerations and procedures for themanipulation, cryopreservation, and long-term storage of HSC sources areknown in the art. See e.g., U.S. Pat. No. 4,199,022; No. 3,753,357; No.4,559,298; No. 5,004,681. There are also various devices with associatedprotocols for the storage of blood. U.S. Pat. No. 6,226,997; No.7,179,643

Considerations in the thawing and reconstitution of HSC sources are alsoknown in the art. U.S. Pat. No. 7,179,643; No. 5,004,681. The HSC sourceblood may also be treated to prevent clumping (see Spitzer, 45 Cancer3075-85 (1980); Stiff et al., 20 Cryobiology 17-24 (1983), and to removetoxic cryoprotective agents (U.S. Pat. No. 5,004,681). Further, thereare various approaches to determining an engrafting cell dose of HSCtransplant units. See U.S. Pat. No. 6,852,534; Kuchler, in BIOCHEM.METHS. CELL CULTURE & VIROLOGY 18-19 (Dowden, Hutchinson & Ross,Strodsburg, Pa., 1964); 10 METHS. MED. RES. 39-47 (Eisen, et al., eds.,Year Book Med. Pub., Inc., Chicago, Ill., 1964).

Diseases of the Hematopoietic System

Hematopoietic progenitor cells or stem cells can be transplanted toregenerate hematopoietic cells in an individual having a disease of thehematopoietic system. Such diseases can include, but are not limited to,cancers (e.g., leukemia, lymphoma), blood disorders (e.g., inheritedanemia, inborn errors of metabolism, aplastic anemia, beta-thalassemia,Blackfan-Diamond syndrome, globoid cell leukodystrophy, sickle cellanemia, severe combined immunodeficiency, X-linked lymphoproliferativesyndrome, Wiskott-Aldrich syndrome, Hunter's syndrome, Hurler's syndromeLesch Nyhan syndrome, osteopetrosis), chemotherapy rescue of the immunesystem, and other diseases (e.g., autoimmune diseases, diabetes,rheumatoid arthritis, system lupus erythromatosis).

Compounds

In one aspect, farnesyl compounds useful for enhancing hematopoieticstem cell engraftment include e.g., S-farnesyl-L-cysteine methyl ester(FCME), and farnesylthioacetic acid (FTA) or derivatives thereof.

In another embodiment, the compound has the structure:

In another embodiment, the compound has the structure:

Other farnesyl compounds amenable to the invention include, but are notlimited to, 2-(farnesylthio)benzoic acid (farnesyl-thiosalicylic acid,FTS), 2-chloro-5-farnesylaminobenzoic acid (NFCB),3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinicacid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate,farnesyl pyrophosphate (FPP), and those described in U.S. Pat. Nos.5,705,528; 5,475,029; European patent No. 356,866; Philips, et. al.,Science (1993), 259: 977-980; Akbar, et al., Proc. Natl. Acad. Sci. USA(1993), 90: 868-872; Tan, et al., J. Biol. Chem., (1991), 26(6):10719-10722 and U.S. Pat. No. 5,705,528.

In one embodiment, two or more of the compounds described herein areadministered together to promote hematopoietic stem cell engraftment. Inanother embodiment, the compound(s) can be administered with a secondagent known to enhance engraftment of hematopoietic stem cells (e.g.,PGE2, BIO etc). In another embodiment, at least one additional agent isused in combination with a compound as described above. In oneembodiment, the additional agent is Prostaglandin E2. In otherembodiments, the additional agent is 16-,16-dimethyl Prostaglandin E2(dmPGE2) (available as FT1050 from FATE THERAPEUTICS™) or BIO. Acombination of agents may be administered simultaneously or separatelyat an interval that permits enhanced hematopoietic stem cellengraftment. For example, an agent that enhances early engraftment(e.g., 0-2 weeks) may be combined with an additional agent that enhanceslonger term engraftment (e.g., <2 weeks). One of skill in the art candetermine an appropriate combination and dosage regime for each agent topermit enhanced engraftment in an individual.

In one embodiment, the hematopoietic stem cells are provided in anadmixture comprising at least one compound as described herein and theadmixture is administered to the subject during the transplantationprocedure.

Zebrafish Competitive Transplant Model

The development of the adult zebrafish whole kidney marrow competitivetransplantation assay described herein provides an important method toquantify hematopoietic stem/progenitor cell engraftment capability aftermarrow transplant, an equivalent of murine bone marrow competitiontransplantation. The conventional zebrafish kidney marrowtransplantation using single marrow from one donor has two drawbacks:First the readout of engraftment requires sacrificing the wild-typerecipients for fluorescence activated cell sorting (FACS) analysis;Second, the big variances among recipients and transplantationprocedures hamper the use of this assay for engraftment quantification.The novel adult zebrafish whole kidney marrow competitivetransplantation assay overcomes these problems and allows directvisualization and quantification of homing and engraftment.

A transparent adult zebrafish, casper, was used as a transplantationrecipient. Casper is a double homozygous pigment mutant for the nacreallele (devoid of melanocytes) and the roy allele (devoid of iridophoresand reduced melanoyctyes), which allows direct visualization of GFPlabeled hematopoietic stem cell homing and engraftment without killingthe recipients. Two marrows are used: Tg(β-actin: GFP) and Red GloFish®respectively to compete with each other at varying ratios e.g., 1:4,which can generate robust competition between the two donors. In oneembodiment, the red marrow is the competitor, as well as an internalcontrol for the transplant. At different timepoints post-transplant, therecipients are anesthetized and two pictures for each recipient aretaken by a fluorescence stereomicroscope with GFP/DsRed filtersindividually. Images are analyzed by software, such as ImageJ, which canquantify the GFP/DsRed fluorescence intensity. The relative engraftmentefficiency of the manipulated green marrow is measured by the ratio ofthe GFP intensity over the DsRed intensity within the same kidney regionafter background subtraction (labeled as G/R). This quantitativeapproach generates results positively correlated with the FACS analysisresults.

Using this assay, it was confirmed that in vitro incubation of the greenmarrow with 10 μM dimethyl-prostaglandin E2 (dmPGE2) or 0.5 μM6-bromorindirubin-3′-oxime (BIO) at room temperature for 2-4 hours canincrease engraftment by 4 weeks post transplant. Furthermore, thezebrafish competitive transplant assay can be used to screen librariesof compounds for those that will enhance engraftment. This chemicalscreen provides a significant advantage over cell culture-based screen(where in vivo analysis is not possible) and murine models (wherelarge-scaled chemical screens are not feasible).

Cell Transplantation

Transplantation of hematopoietic cells has become the treatment ofchoice for a variety of inherited or malignant diseases. While earlytransplantation procedures utilized the entire bone marrow (BM)population, recently, more defined populations, enriched for stem cells(CD34 cells) have been used. In addition to the marrow, such cells couldbe derived from other sources such as bone marrow stem cells mobilizedto the peripheral blood (PB) and neonatal umbilical cord blood (CB).

The donor and the recipient can be a single individual or differentindividuals, for example, autologous or allogeneic transplants,respectively. When allogeneic transplantation is practiced, regimes forreducing implant rejection and/or graft vs. host disease, as well knownin the art, should be undertaken. Such regimes are currently practicedin human therapy. The cell populations selected can also be depleted ofT lymphocytes, which may be useful in the allogeneic and haploidenticaltransplants setting for reducing graft-versus-host disease.

Most advanced regimes are disclosed in publications by Slavin S. et al.,e.g., J Clin Immunol 2002; 22:64, and J Hematother Stem Cell Res 2002;11:265, Gur H. et al. Blood 2002; 99:4174, and Martelli M F et al, SeminHematol 2002; 39:48, which are incorporated herein by reference.

In another embodiment of the invention, INPROL can be employed in amethod for preparing autologous hematopoietic cells for transplantation,as described in U.S. Pat. No. 7,115,267, which is herein incorporated byreference in its entirety. The hematopoietic cells are treated ex vivowith an effective amount of INPROL to inhibit stem cell division andthen purged of cancerous cells by administering to the marrow culturesan effective amount of a chemotherapeutic agent or radiation.Chemotherapy agents with specificity for cycling cells are preferred.Marrow thus treated is re-injected into the autologous donor.Optionally, the patient is treated with stem cell stimulatory amounts ofINPROL and/or another agent known to stimulate hematopoiesis to improvethe hematopoietic reconstitution of the patient. Such a technique allowsfor effective purging of tumor cells during autologous bone marrowgrafts while protecting hematopoietic stem cells. Such protection can beafforded with either ex vivo or in vivo purging protocols. Oncesuccessfully transplanted, there is a need for stem cells to rapidlyproliferate to regenerate normal bone marrow function. This can beafforded by the use of INPROL at stem cell stimulatory amounts whichstimulates cycling of stem cells and enhances recovery of bone marrowfunction.

Methods for Administering Cells

Stem cells can be administered to a subject either locally orsystemically. Methods for administering bone marrow transplants to asubject are known in the art and are described in medical textbooks,e.g., Whedon, M. B. (1991) Whedon, M. B. “Bone Marrow Transplantation:Principles, Practice, and Nursing Insights”, Boston:Jones and BartlettPublishers. In certain embodiments, bone marrow cells from a healthypatient can be removed, preserved, and then replicated and re-infusedshould the patient develop an illness which either destroys the bonemarrow directly or whose treatment adversely affects the marrow. If thepatient is receiving his or her own cells, this is called an autologoustransplant; such a transplant has little likelihood of rejection.

Exemplary methods of administering stem cells to a subject, particularlya human subject, include injection or transplantation of the cells intotarget sites in the subject. The hematopoietic stem cells and/orhematopoietic progenitor cells can be inserted into a delivery devicewhich facilitates introduction, by injection or transplantation, of thecells into the subject. Such delivery devices include tubes, e.g.,catheters, for injecting cells and fluids into the body of a recipientsubject. In a preferred embodiment, the tubes additionally have aneedle, e.g., a syringe, through which the cells of the invention can beintroduced into the subject at a desired location. The stem cells can beinserted into such a delivery device, e.g., a syringe, in differentforms. For example, the cells can be suspended in a solution, oralternatively embedded in a support matrix when contained in such adelivery device.

Support matrices in which the stem cells can be incorporated or embeddedinclude matrices which are recipient-compatible and which degrade intoproducts which are not harmful to the recipient. The support matricescan be natural (e.g. collagen etc.) and/or synthetic biodegradablematrices. Synthetic biodegradable matrices include synthetic polymerssuch as polyanhydrides, polyorthoesters, and polylactic acid; see also,for example, U.S. Pat. No. 4,298,002 and U.S. Pat. No. 5,308,701.

As used herein, the term “solution” includes a pharmaceuticallyacceptable carrier or diluent in which the cells of the invention remainviable. Pharmaceutically acceptable carriers and diluents includesaline, aqueous buffer solutions, solvents and/or dispersion media. Theuse of such carriers and diluents is known in the art. The solution ispreferably sterile and fluid to the extent that easy syringabilityexists.

Preferably, the solution is stable under the conditions of manufactureand storage and preserved against the contaminating action ofmicroorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobu-tanol, phenol, ascorbic acid, thimerosal,and the like. Solutions of the invention can be prepared byincorporating stem cells as described herein in a pharmaceuticallyacceptable carrier or diluent and, as required, other ingredientsenumerated above, followed by filtered sterilization.

Dosage and Administration

In one aspect, the methods described herein provide a method forenhancing engraftment of hematopoietic stem and/or progenitor cellsfollowing a bone marrow transplant in a subject. In one embodiment, thesubject can be a mammal. In another embodiment, the mammal can be ahuman, although the invention is effective with respect to all mammals.The method comprises administering to the subject an effective amount ofa pharmaceutical composition comprising a farnesyl compound such asS-farnesyl-L-cysteine methyl ester (FCME), or farnesylthioacetic acid(FTA) in a pharmaceutically acceptable carrier. The dosage range for thecompound depends upon the potency, and are amounts large enough toproduce the desired effect e.g., an increase in the efficiency and/orrate of hematopoietic progenitor cell or stem cell engraftment. Thedosage should not be so large as to cause adverse side effects.

Generally, the dosage will vary with the particular compound used, andwith the age, condition, and sex of the patient. The dosage can bedetermined by one of skill in the art and can also be adjusted by aphysician in the event of any complication. Typically, the dose willrange from 0.001 mg/kg body weight to 5 g/kg body weight. In someembodiments, the dose will range from 0.001 mg/kg body weight to 1 g/kgbody weight, from 0.001 mg/kg body weight to 0.5 g/kg body weight, from0.001 mg/kg body weight to 0.1 g/kg body weight, from 0.001 mg/kg bodyweight to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kgbody weight, from 0.001 mg/kg body weight to 10 mg/kg body weight, from0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001 mg/kg bodyweight to 1 mg/kg body weight, from 0.001 mg/kg body weight to 0.1 mg/kgbody weight, from 0.001 mg/kg body weight to 0.005 mg/kg body weight.Alternatively, in some embodiments the dose range is from 0.1 g/kg bodyweight to 5 g/kg body weight, from 0.5 g/kg body weight to 5 g/kg bodyweight, from 1 g/kg body weight to 5 g/kg body weight, from 1.5 g/kgbody weight to 5 g/kg body weight, from 2 g/kg body weight to 5 g/kgbody weight, from 2.5 g/kg body weight to 5 g/kg body weight, from 3g/kg body weight to 5 g/kg body weight, from 3.5 g/kg body weight to 5g/kg body weight, from 4 g/kg body weight to 5 g/kg body weight, from4.5 g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight to5 g/kg body weight. In one embodiment, the dose range is from 5 μg/kgbody weight to 30 μg/kg body weight.

Alternatively, the dose range will be titrated to maintain serum levelsbetween 5 μg/mL and 30 μg/mL.

A compound as described herein can be given once a day, less than once aday, multiple times a day, or continuously in order to achieve atherapeutically effective dose. Administration of the doses recitedabove can be repeated for a limited period of time. In a preferredembodiment, the doses recited above can be administered daily forseveral weeks or months. The duration of treatment depends upon thesubject's clinical progress and responsiveness to therapy. A“therapeutically effective amount” is an amount of a compound that issufficient to produce a measurable change in engraftment efficiency orrate (see “Efficacy Measurement” below). Such effective amounts can begauged in clinical trials as well as animal studies.

Compounds useful with the methods described herein can be administeredintravenously, intranasally, orally, by inhalation, intraperitoneally,intramuscularly, subcutaneously, intracavity, and can be delivered byperistaltic means, if desired, or by other means known by those skilledin the art. In one embodiment the compounds used herein are administeredorally, or intravenously to a patient following hematopoietic progenitorcell transplantation. The compound can be administered intravenously byinjection or by gradual infusion over time.

In some embodiments, a farnesyl compound (e.g., S-farnesyl-L-cysteinemethyl ester (FCME), or farnesylthioacetic acid (FTA)) is administeredas part of a combination therapy regime. Therapeutic compositionscontaining at least one compound as described herein can beconventionally administered in a unit dose form. The term “unit dose”when used in reference to a therapeutic composition refers to physicallydiscrete units suitable as unitary dosage for the subject, each unitcontaining a predetermined quantity of active material calculated toproduce the desired therapeutic effect in association with the requiredphysiologically acceptable diluent, i.e., carrier, or vehicle. Acombination of more than one agent can be administered in one or morepharmaceutical compositions (i.e., together in a unit dose, such as apill or tablet, or as two separate compositions). In one embodiment, theagents are administered separately and can be administered in an order,or at an interval of time that provides effective hematopoietic stemcell graft enhancement as directed by one of skill in the art ofmedicine. In addition, the agents can be administered using the same ordifferent modes of administration.

The compositions of a combination therapy are administered in a mannercompatible with the dosage formulation, and in a therapeuticallyeffective amount. The quantity to be administered and timing depends onthe subject to be treated, capacity of the subject's system to utilizethe active ingredient, and degree of therapeutic effect desired. Preciseamounts of each active ingredient required to be administered depend onthe judgment of the practitioner and are particular to each individual.However, suitable dosage ranges for systemic application are disclosedherein and depend on the route of administration. Suitable regimes foradministration are also variable, but are typified by an initialadministration followed by repeated doses at one or more hour intervalsby a subsequent injection or other administration. Alternatively,continuous intravenous infusion sufficient to maintain concentrations inthe blood in the ranges specified for in vivo therapies arecontemplated.

Pharmaceutical Compositions

The present invention involves therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions contain a physiologically tolerable carrier together withan active compound as described herein, dissolved or dispersed thereinas an active ingredient. In a preferred embodiment, the therapeuticcomposition is not immunogenic when administered to a mammal or humanpatient for therapeutic purposes, unless so desired. As used herein, theterms “pharmaceutically acceptable”, “physiologically tolerable” andgrammatical variations thereof, as they refer to compositions, carriers,diluents and reagents, are used interchangeably and represent that thematerials are capable of administration to or upon a mammal without theproduction of undesirable physiological effects such as nausea,dizziness, gastric upset and the like. A pharmaceutically acceptablecarrier will not promote the raising of an immune response to an agentwith which it is admixed, unless so desired. The preparation of apharmacological composition that contains active ingredients dissolvedor dispersed therein is well understood in the art and need not belimited based on formulation. Typically such compositions are preparedas injectable either as liquid solutions or suspensions, however, solidforms suitable for solution, or suspensions, in liquid prior to use canalso be prepared. The preparation can also be emulsified or presented asa liposome composition. The active ingredient can be mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient and in amounts suitable for use in the therapeuticmethods described herein. Suitable excipients are, for example, water,saline, dextrose, glycerol, ethanol or the like and combinationsthereof. In addition, if desired, the composition can contain minoramounts of auxiliary substances such as wetting or emulsifying agents,pH buffering agents and the like which enhance the effectiveness of theactive ingredient. The therapeutic composition of the present inventioncan include pharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in theinvention that will be effective in the treatment of a particulardisorder or condition will depend on the nature of the disorder orcondition, and can be determined by standard clinical techniques.

Efficacy measurement

Assessing Engraftment

Engraftment after lethal ablation of the bone marrow can be assessed bymeasuring hematopoietic blood cell counts; in particular white bloodcell counts. Following lethal ablation, recovery of normal white bloodcell counts is a functional measure of successful engraftment. In aclinical context, this can be accompanied by the measurement ofcellularity in the bone marrow through serial bone marrowpunctions/biopsies and/or by human leukocyte antigen (HLA) typing ofcirculating white blood cells. Bone marrow aspirates can also beassessed for donor chimerism as a measure of engraftment.

All blood cell types can be indicative of engraftment, but depending ontheir half lives, provide a more or less sensitive measure ofengraftment. Neutrophils have a very short half life (just hours in theblood), and thus are a very good measure of early engraftment. Plateletsalso have a short half life, but they are usually the last blood elementto recover to pre-transplant levels, which may not make them suitable asa marker of early engraftment.

Thus, it is noted herein that cells useful for determining engraftmentof hematopoietic progenitor cells are those that recover relativelyrapidly following transplantation and have a relatively short half-life(e.g., neutrophils). In one embodiment of the methods described herein,hematopoietic progenitor cell engraftment is assessed by detectingand/or measuring the level of recovery of neutrophils in an individual.

Efficacy

The efficacy of a given treatment to enhance hematopoietic cellengraftment can be determined by the skilled clinician. However, atreatment is considered “effective treatment,” as the term is usedherein, if any one or all of the signs or symptoms of e.g., poorhematopoietic cell engraftment are altered in a beneficial manner, otherclinically accepted symptoms are improved, or even ameliorated, e.g., byat least 10% following treatment with a compound as described herein.Efficacy can also be measured by a failure of an individual to worsen asassessed by hospitalization, need for medical interventions (i.e.,progression of the disease is halted), or incidence of engraftmentfailure. Methods of measuring these indicators are known to those ofskill in the art and/or are described herein. Treatment includes anytreatment of a disease in an individual or an animal (some non-limitingexamples include a human, or a mammal) and includes: (1) inhibiting thedisease, e.g., preventing engraftment failure; or (2) relieving thedisease, e.g., causing regression of symptoms. An effective amount forthe treatment of a disease means that amount which, when administered toa mammal in need thereof, is sufficient to result in effective treatmentas that term is defined herein, for that disease. Efficacy of an agentcan be determined by assessing physical indicators of, for examplehematopoietic cell engraftment, such as e.g., neutrophil production,white blood cell count, hematopoietic cell numbers, presence/absence ofanemia etc. Efficacy can be assessed in animal models of bone marrowtransplantation, for example treatment of a rodent following bone marrowtransplantation, and any treatment or administration of the compositionsor formulations that leads to an increase of at least one symptom ofhematopoietic cell engraftment.

Kits

Also provided herein are kits comprising components for (a) ex vivotreatment of isolated hematopoietic stem cells, (b) preparing admixturesof isolated HSC and a compound as described herein, and (c)administration of a compound as described herein to a subject.

In one embodiment, provided herein is a pack or kit comprising one ormore containers filled with one or more of the compounds describedherein described herein in the ‘Compounds’ section. Thus, for example, akit described herein comprises one or more farnesyl compounds asdescribed herein. Such kits optionally comprise solutions and buffers asneeded or desired. The kit optionally includes containers orcompositions for making an expanded population of isolated HSC.Optionally associated with such pack(s) or kit(s) are instructions foruse and packaging materials therefor. In one embodiment, the kit furthercomprises additional agent such as prostaglandin E2, 16-,16-dimethylProstaglandin E2 (dmPGE2) (available as FT1050 from FATE THERAPEUTICS™),and/or BIO.

Also provided is a kit for providing an effective amount of a compoundas described herein to enhance engraftment by administering to a subjectand comprising one or more doses of at least one compound as describedherein for use over a period of time, wherein the total number of dosesof the at least one compound in the kit equals the effective amount ofthe compound or combination thereof sufficient to enhance engraftment ina subject. The period of time is from about one to several days or weeksor months. Thus, the period of time is from at least about five, six,seven, eight, ten, twelve, fourteen, twenty, twenty-one or thirty daysor more or any number of days between one and thirty. The doses of theat least one compound can be administered once, twice, three times ormore daily or weekly. The kit provides one or multiple doses for atreatment regimen.

A kit for providing an effective amount of at least one compound asdescribed herein for treating isolated HSCs is described. The kitcomprises one or more aliquots of at least one compound or combinationsthereof for administration to HSC or a mixture of HSC and HSC-supportingcells over a period of time, wherein the aliquots equal the effectiveamount of the compounds required to expand the population of HSC. Theperiod of time is from about one to several hours or one to severaldays. The amount of the compound or combination thereof is administeredonce, twice, three times or more daily or weekly and the kit providesone or multiple aliquots.

Optionally, the methods and kits comprise effective amounts of at leastone compound for administering to the subject to effect enhancedengraftment in a second or subsequent regime for a specific period oftime. The second or subsequent period of time, like the first period oftime, is, for example, at least one or more days, weeks or months, suchas, for example, at least four, five, six, seven, eight, nine, ten,eleven, twelve, fourteen, twenty one, or thirty days or any number ofdays between. In the methods herein, the interval between the firsttreating period and the next treating period is optionally, for example,days, weeks, months or years. Thus, the interval between the firstperiod of time and the next period of time is, for example, at leastfour, five, six, seven, eight, nine, ten, eleven, twelve, fourteen,twenty one, or twenty eight days or in number of days between. Thistreating schedule is repeated several times or many times as necessary.Such schedules are designed to correlate with repeated bone marrowdepleting events such as repeated chemotherapy treatments or radiationtherapy treatments. Optionally, a drug delivery device or componentthereof for administration is included in a kit. Disclosed are materialsor steps in a method, compositions, and components that are used for,are used in conjunction with, are used in preparation for, or areproducts of the disclosed method and compositions. These and othermaterials are disclosed herein, and it is understood that whencombinations, subsets, interactions, groups, etc. of these materials orsteps are disclosed that, while specific reference of each variousindividual and collective combinations and permutation of thesematerials or steps may not be explicitly disclosed, each is specificallycontemplated and described herein.

It is understood that the foregoing detailed description and thefollowing examples are illustrative only and are not to be taken aslimitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments, which will be apparent tothose of skill in the art, may be made without departing from the spiritand scope of the present invention. Further, all patents, patentapplications, and publications identified are expressly incorporatedherein by reference for the purpose of describing and disclosing, forexample, the methodologies described in such publications that might beused in connection with the present invention. These publications areprovided solely for their disclosure prior to the filing date of thepresent application. Nothing in this regard should be construed as anadmission that the inventors are not entitled to antedate suchdisclosure by virtue of prior invention or for any other reason. Allstatements as to the date or representation as to the contents of thesedocuments are based on the information available to the applicants anddo not constitute any admission as to the correctness of the dates orcontents of these documents.

The present invention may be as defined in any one of the followingnumbered paragraphs.

-   1. A method for enhancing hematopoietic cell engraftment in a    subject, the method comprising:

(a) contacting a population of hematopoietic cells with a compoundselected from the group consisting of: S-farnesyl-L-cysteine methylester (FCME), farnesylthioacetic acid (FTA), 2-(farnesylthio)benzoicacid (farnesyl-thiosalicylic acid, FTS), 2-chloro-5-farnesylaminobenzoicacid (NFCB), 3-(farnesylthio)pyridine-2-carboxylic acid (farnesylthionicoatinic acid, FTN), (farnesylthio)propanoic acid (FTP), farnesylacetate, and farnesyl pyrophosphate (FPP); and

(b) administering treated cells from step (a) to a subject in needthereof, wherein the engraftment of the hematopoietic cells treated withthe compound is increased compared to engraftment of a population ofuntreated hematopoietic cells.

-   2. The method of paragraph 1, wherein the compound is    S-farnesyl-L-cysteine methyl ester (FCME).-   3. The method of paragraph 1 or 2, wherein the compound has a    structure of

-   4. The method of any one of the preceding paragraphs, wherein the    compound is farnesylthioacetic acid (FTA).-   5. The method of any one of the preceding paragraphs, wherein the    compound has a structure of

-   6. The method of any one of the preceding paragraphs, wherein the    population of hematopoietic cells is derived from cord blood.-   7. The method of any one of the preceding paragraphs, wherein the    population of hematopoietic cells is derived from bone marrow.-   8. The method of any one of the preceding paragraphs, wherein the    population of hematopoietic cells is derived from blood.-   9. The method of any one of the preceding paragraphs, wherein the    population of hematopoietic cells are isolated cells.-   10. The method of any one of the preceding paragraphs, wherein the    population of hematopoietic cells is a heterogeneous or homogeneous    population of cells.-   11. The method of any one of the preceding paragraphs, wherein the    contacting step is performed on ex vivo cells in culture.-   12. The method of any one of the preceding paragraphs, wherein the    subject is a human subject.-   13. The method of any one of the preceding paragraphs, wherein the    hematopoietic cells are hematopoietic stem cells.-   14. The method of any one of the preceding paragraphs, wherein the    hematopoietic cells are hematopoietic progenitor cells.-   15. A method for enhancing hematopoietic cell engraftment in a    subject following hematopoietic cell transplantation, the method    comprising: administering to a subject following hematopoietic cell    transplantation a therapeutically effective amount of a compound    selected from the group consisting of: S-farnesyl-L-cysteine methyl    ester (FCME), farnesylthioacetic acid (FTA), 2-(farnesylthio)benzoic    acid (farnesyl-thiosalicylic acid, FTS),    2-chloro-5-farnesylaminobenzoic acid (NFCB),    3-(farnesylthio)pyridine-2-carboxylic acid (farnesyl thionicoatinic    acid, FTN), (farnesylthio)propanoic acid (FTP), farnesyl acetate,    and farnesyl pyrophosphate (FPP),

wherein the level of engraftment of the hematopoietic cells in thesubject treated with S-farnesyl-L-cysteine methyl ester (FCME), and/orfarnesylthioacetic acid (FTA) is increased compared to engraftment of apopulation of hematopoietic cells in an untreated subject.

-   16. The method of paragraph 15, wherein the compound is    S-farnesyl-L-cysteine methyl ester (FCME).-   17. The method of paragraph 15 or 16, wherein the compound has a    structure of

-   18. The method of any one of paragraphs 15-17, wherein the compound    is farnesylthioacetic acid (FTA).-   19. The method of any one of paragraphs 15-18, wherein the compound    has a structure of

-   20. The method of any one of paragraphs 15-19, wherein the    population of hematopoietic cells is derived from cord blood.-   21. The method of any one of paragraphs 15-20, wherein the    population of hematopoietic cells is derived from bone marrow.-   22. The method of any one of paragraphs 15-21, wherein the    population of hematopoietic cells is derived from blood.-   23. The method of any one of paragraphs 15-22, wherein the    population of hematopoietic cells are isolated cells.-   24. The method of any one of paragraphs 15-23, wherein the    population of hematopoietic cells is a heterogeneous or homogeneous    population of cells.-   25. The method of any one of paragraphs 15-24, wherein the subject    is a human subject.-   26. The method of any one of paragraphs 15-25, wherein the    hematopoietic cells are hematopoietic stem cells.-   27. The method of any one of paragraphs 15-26, wherein the    hematopoietic cells are hematopoietic progenitor cells.-   28. The method of any one of the preceding paragraphs, further    comprising administering at least one additional agent that enhances    engraftment of hematopoietic stem cells.-   29. The method of any one of the preceding paragraphs, wherein the    at least one additional agent comprises PGE2 or BIO.

EXAMPLES Example 1 Zebrafish Competitive Transplant Assay Development

Described herein is an adult zebrafish competitive marrowtransplantation assay, which can be used to directly visualize andquantify engraftment, and measure the migration, self-renewal,proliferation and differentiation properties of hematopoietic stem cells(HSCs) in zebrafish. A chemical screen with the zebrafish WKMcompetitive transplantation model accelerates the discovery of novelpathways and signaling networks regulating these properties of HSCs. Thezebrafish embryonic and adult hematopoietic models also help tocharacterize and understand the mechanism of the pathways. Last but notleast, compared with traditional genetic methods, chemical geneticapproaches provide not only pathways relevant to the biological process,but also small molecules as the starting point of drug development toincrease the HSC engraftment capability and improve the engraftmentefficiency of human HSC transplantation.

A double-color labeled competitive transplantation assay was developed,as shown by the flow chart in FIG. 1A. GFP+ and DsRed2+ marrow cells areisolated from Tg(β-actin:GFP) and commercially available Red GloFish®.The two donor populations are mixed at a certain ratio and injected intosublethally irradiated casper recipients retro-orbitally. The DsRed2+marrow is used as the competitor. Four weeks later, the engraftment isvisualized by fluorescence dissection scope. Pictures of GFP and DsRedengraftment are taken for each recipient and analyzed by ImageJ. Thekidney region is manually selected and the average fluorescenceintensities of both green and red are measured within the same region.The average background intensity is measured in a region outside thefish, and subtracted from the kidney intensity. The calculation of therelative engraftment is shown by the equation in FIG. 1B.

To test whether the competitive transplantation assay can faithfully andsensitively detect changes of the donor cell ratios, the donor G:R(green:red) ratios were increased from 1:3 to 1:2 to 1:1, while keepingthe total number of donor cells constant as 200,000. The readout at 4wpt (week post transplant) shows an increased G/R by increasing thedonor G:R ratio (FIG. 2). The 1:3 group was set as the control, and itsaverage G/R plus two standard deviation as the cutoff line. Recipientswith a G/R above this line are called positive, which means their greenmarrows have a competitive advantage than the control. By increasing thedonor G:R ratio, the percentage of positive recipients also increased asshown in the table in FIG. 2B. This result proves the assay canfaithfully represent the donor ratio and sensitively detect the ratiochanges.

The applicability of the zebrafish model was confirmed by testing twoknown signaling pathways regulating HSCs using a chemical geneticapproach. Two compounds: BIO and dmPGE2 were chosen. BIO is a GSK-3inhibitor and thus activates the Wnt pathway. dmPGE2 is a stabilizedderivative of prostaglandin E2, which binds to the PGE2 receptor. Bothpathways increase HSC self-renewal. After cell preparation, the greenmarrow cells were incubated with DMSO, BIO or dmPGE2 in vitro for 4 hrs,mixed with freshly isolated red marrow cells at a ratio of 1:4 andinjected into casper. At 4 wpt, both the average G/R and the percentageof positive recipients in the BIO/dmPGE2 treated groups weresignificantly increased compared with a DMSO control (FIG. 3C). Theresults are very similar to what has been observed in mouse competitivetransplant.

To explore the feasibility of using this competitive transplantationassay for a chemical screen, as well as the optimal timepoint forreadout, small-scaled pilot screen was performed with 10 bioactivecompounds. We started with 10 recipients per treatment, and followed theengraftment every week from 2 weeks post-transplant (wpt) to 4 wpt. Theengraftment signal was not strong enough to observe the effect of mostchemical treatments at 2 wpt (data not shown). By four weeks, dmPGE2 andBIO have an enhanced signal above the rest of the chemicals tested. Thisindicates that although stem cell homing, lodgement, and engraftmenthave taken place by 2 weeks, the amplification of hematopoiesis between2-4 weeks allows a more sensitive detection of the success ofenhancement of these biological processes, particularly for a screen. Inlater timepoints, recipient survival dropped sharply due to multiplereasons. In one embodiment, the 4 wpt is the optimal timepoint for thescreen.

An Exemplary Competitive Transplantation Protocol.

Adult zebrafish donors are anaesthetized with 0.2% tricaine before bloodand kidney collection. Peripheral blood is obtained from adult casper bycardiac puncture with micropipette tips coated with heparin andcollected into 0.9×PBS containing 5% FCS. About 3-5 million red bloodcells are harvested from one donor. To dissect whole kidney marrow(WKM), a ventral midline incision is made on the donor fish. Wholekidney is dissected out and placed into ice-cold 0.9×PBS containing 5%FCS. Single-cell suspensions are generated by aspiration followed byfiltering through a 40-m nylon mesh filter into a 50 ml conical tube.The flow-though part is diluted with a final volume of 25 ml andcentrifuged at 1,500 rpm for 8 minutes. The supernatant is discarded andthe pellet cells are resuspended in 1 ml 0.9×PBS containing 5% FCS. Thenumber of viable cells are counted with a hemocytometer. On average,500,000-700,000 cells can be harvested per donor. Finally, theperipheral blood and kidney marrow cells are centrifuged at 1,500 rpmfor 8 minutes, resuspended in 0.9×PBS containing 5% FCS and mixed at acertain ratio and concentration. Casper recipients are anesthetized intricaine. 4 μl of the cell suspension mixture described above isinjected into the circulation retro-orbitally through a Hamilton syringe(26 s gauge, 10 μl volume). The retro-orbital injection is much moreconsistent than the intra-cardiac injection previously used.Transplanted recipients can be anesthetized in tricaine and visualizedover time on a Zeiss Discovery V8 stereomicroscope with a 1.2× PlanApolens and GFP/DsRed filters. Images are captured using AxioVisionsoftware and for each recipient, two images are taken with GFP or DsRedfilter respectively. The images are all saved in .tif format andanalyzed using ImageJ software. Kidney region is manually selected forevery fish and the average fluorescence intensities per pixel of bothGFP and DsRed are measured within the same region. Background for eachimage is measured in a region outside the fish, and subtracted from thecorrelated kidney fluorescence intensity. The relative engraftmentefficiency (G/R) for every single recipient is measured by the ratio ofGFP intensity (background subtracted) and DsRed intensity (backgroundsubtracted).

One of the advantages of the casper fish is that the kidney can bevisualized as a solid organ. This establishes a stem cell niche based onlocalized fluorescence intensity. The ratio of green to red marrow cellsis well correlated between the ImageJ fluorescence intensity and FACSanalysis. As a screening technology, fluorescence intensity in thekidney has great advantages since the animal does not have to besacrificed for FACS analysis and can be followed longitudinally. Inaddition, the green to red intensity is positively correlated to theFACS results. Even visual examination of green vs. red under afluorescent dissecting scope reveals competitive engraftment.

With the competitive transplant assay established, it was evaluatedwhether chemicals of known biologic action in stem cells could affectengraftment in zebrafish. An experimental approach for the competitivetransplant assay is outlined herein in FIG. 4. A variety of differenttimes from 2 weeks to 3 months after transplantation were studied. The4-week time period is more amenable to high-throughput screens thanwaiting the 3 months for long-term reconstitution. Although the fish canbe evaluated at 3 months, for screening purposes a chemical that wouldallow enhanced engraftment at 4 weeks and also at 3 months is preferred.One such chemical is dmPGE2, which enhances hematopoietic self-renewalin mouse, and BIO, a chemical that inhibits GSK-3, leads to enhancementof the wnt pathway and also increases self-renewal. Both of thesechemicals score positive in the competitive transplant model in thezebrafish at 4 weeks and at 3 months.

The Chemical Screen.

The chemical screen utilizes β-actin GFP whole kidney marrow competedagainst red glow fish whole kidney marrow. 20,000 GFP positive cells arecompeted against 80,000 DsRed positive marrow cells. In thistest:competitor ratio, low levels of GFP expressing marrow at 4 weeks isevident. The cells are incubated in the chemicals for 4 hours beforetransplantation and washed off. 10 recipients are injected with thetreated cells at day 2 after the irradiation and by 4 weeks, therecipient casper fish is visualized and the fluorescence intensity ratioof green vs. red is calculated. Recipients in which an increasedintensity with the green compared to red is observed indicates achemical that enhances engraftment, perhaps by affecting homing,engraftment, or self-renewal.

β-actin: GFP (whole kidney marrow) WKM cells are resuspended in 0.9×PBScontaining 5% FCS at a concentration of 1,000/1 and aliquoted into a96-well sterilized clear round-bottom tissue culture plate (200μl/well). Compounds from ICCB Known Bioactive Library are added intoeach well (1:200 dilution), and the cells are incubated with compoundsat room temperature for 4 hrs before being centrifuged at 1,500 rpm for8 minutes. 800 k WKM cells from Red GloFish and 1,600 k peripheral bloodcells are added into each well to make the final volume in each well tobe 401. The cells in each well are transplanted into 10 recipients. Therelative engraftment efficiency is evaluated at 4 week post transplant.

Initially, a small pilot screen using a panel of ten bioactive chemicalswas screened and confirmed the optimal time for evaluation (FIG. 6). Twoweeks after the transplant, there was very little engraftment observablebased treatment with chemicals. By four weeks, dmPGE2 and BIO have anenhanced signal above the rest of the chemicals tested.

The throughput that can be accomplished is one screen per day andtesting 20 chemicals per day. This would mean that 10 recipient fish areused for each chemical assayed and these fish will be followed for 2.5to 3 months. To do the screen, 12 green fish and 45 red fish would beneeded as donors and these should be old enough and of sufficient sizeto provide sufficient marrow cells for the screen. In terms of anexperimental day, 1.5 hours of time is needed for GFP positive marrowpreparation and then the cells are treated with chemicals for 4 hours.Then red marrow preparation is done for another 1.5 hours and about 1.5hours is required for transplantation of 220 fish. This library consistsof 500 chemicals that have been selected for their known biologicalactivity.

Example 2 Exemplary Compounds that Enhance Engraftment

240 compounds have been screened using the Zebrafish competitivetransplant assay described herein above. Twenty compounds showed apositive effect on engraftment in the primary screen. Ten out the twentycompounds have been repeated in the second round of the screen. Farnesylcompounds were found to have a positive effect on hematopoietic stemand/or progenitor cell engraftment as shown in Table 1:

TABLE 1 Exemplary farnesyl compounds that enhance engraftment in azebrafish model compound name conc. mechanism PvalueS-Farnesyl-L-cysteine 5 mM Bioactive lipids MDR 0.04 methyl ester (FCME)ATPase activator Farnesylthioacetic 5 mM Bioactive lipids 0.046 acid(FTA) Carboxymethylation inhibitor

Five positive compounds from the Zebrafish screen were further confirmedusing a mouse whole bone marrow competitive transplantation assay.20,000 CD45.1 donor bone marrow cells treated with different compoundsat 37° C. for 3 hours, to compete with 200,000 CD45.2 bone marrow cells.The peripheral blood from each recipient are collected at different timepoints: 3 weeks post transplant (wpt), 6 wpt, and 12 wpt. The lineagecontribution from CD45.1 donor marrows are analyzed by FACS. The 3 wptand 6 wpt results are shown herein in FIG. 7. This experiment confirmedthe drug effects of increasing marrow repopulation in mammals.

TABLE 2 Farnesyl compounds confirmed to enhance hematopoietic cellengraftment in a mouse model. compound name conc. mechanism PvalueS-Farnesyl-L-cysteine 5 mM Bioactive lipids MDR 0.04 methyl ester ATPaseactivator

1. A method for enhancing hematopoietic cell engraftment in a subjectfollowing hematopoietic cell transplantation, the method comprising:administering to a subject following hematopoietic cell transplantationa therapeutically effective amount of a compound selected from the groupconsisting of: S-farnesyl-L-cysteine methyl ester (FCME), andfarnesylthioacetic acid (FTA), wherein the level of engraftment of saidhematopoietic cells in said subject treated with S-farnesyl-L-cysteinemethyl ester (FCME), and/or farnesylthioacetic acid (FTA) is increasedcompared to engraftment of a population of hematopoietic cells in anuntreated subject.
 2. The method of claim 15, wherein the compound isS-farnesyl-L-cysteine methyl ester (FCME).
 3. The method of claim 1,wherein the compound is farnesylthioacetic acid (FTA).
 4. The method ofclaim 1, wherein said population of hematopoietic cells is derived fromcord blood.
 5. The method of claim 1, wherein said population ofhematopoietic cells is derived from bone marrow.
 6. The method of claim1, wherein said population of hematopoietic cells is derived from blood.7. The method of claim 1, wherein said population of hematopoietic cellsare isolated cells.
 8. The method of claim 1, wherein said population ofhematopoietic cells is a heterogeneous or homogeneous population ofcells.
 9. The method of claim 1, wherein said contacting step isperformed on ex vivo cells in culture.
 10. The method of claim 1,wherein said subject is a human subject.
 11. The method of claim 1,wherein said hematopoietic cells are hematopoietic stem cells.
 12. Themethod of claim 1, wherein said hematopoietic cells are hematopoieticprogenitor cells.
 13. A method for enhancing hematopoietic cellengraftment in a subject, the method comprising: (a) contacting apopulation of hematopoietic cells with a compound selected from thegroup consisting of: 2-chloro-5-farnesylaminobenzoic acid (NFCB),(farnesylthio)propanoic acid (FTP), farnesyl acetate, and farnesylpyrophosphate (FPP); and (b) administering treated cells from step (a)to a subject in need thereof, wherein the engraftment of saidhematopoietic cells treated with said compound is increased compared toengraftment of a population of untreated hematopoietic cells.
 14. Themethod of claim 13, wherein said population of hematopoietic cells isderived from cord blood.
 15. The method of claim 13, wherein saidpopulation of hematopoietic cells is derived from bone marrow or fromblood.
 16. The method of claim 13, wherein said population ofhematopoietic cells are isolated cells.
 17. The method of claim 13,wherein said population of hematopoietic cells is a heterogeneous orhomogeneous population of cells.
 18. The method of claim 13, whereinsaid contacting step is performed on ex vivo cells in culture.
 29. Themethod of claim 13, wherein said subject is a human subject.
 20. Themethod of claim 13, wherein said hematopoietic cells are hematopoieticstem cells or are hematopoietic progenitor cells.